Monoclonal antibodies able to neutralize HIV-1 primary isolates of multiple genetic subtypes are sought to identify target epitopes for inclusion in an effective HIV-1 vaccine. Current techniques for generating human monoclonal antibodies include hybridoma fusions, EBV-immortalization, and combinatorial libraries of Ig heavy and light chains prepared from leukocytes of seropositive donors. The antibodies produced using these techniques differ systematically from each other, suggesting that each technique imposes selection biases which restrict the pool of clonable immunoglobulins (lgs). Such restrictions, as yet uncharacterized, could contribute to the historic difficulty in generating human mAbs recognizing conserved neutralization epitopes. In any case, these established techniques have proven extremely inefficient in generating antibodies with the requisite neutralization breadth and potency. Recent technical advances facilitate the use of an alternative approach that should overcome these limitations. Inverse immunohistology, the application of a labeled antigen to identify the site of cognate antibodies in situ, can identify in tissue sections those B cells producing antibodies that recognize HIV-1 antigens. Inverse immunohistology has been combined with mechanical microdissection to facilitate molecular analyses of Igs and defined antigenic activities. With the advent of laser capture microdissection, a technique that greatly increases the speed and ease of ell recovery from tissue sections, these powerful techniques now can readily be applied to the cloning of Igs recognizing HIV-1 antigens and suitable for production of recombinant Fabs or mAbs. We will survey lymphoid tissues from macaques infected with chimeric simian-human immunodeficiency viruses (SHIV) bearing envelopes derived from primary HIV-1 isolates, and identify B cells producing HIV-1 specific antibodies in situ. In order to identify B cells producing antibodies with desirable binding characteristics, we will employ a panel of oligomeric HIV-1 envelopes derived from primary isolates of multiple genetic subtypes in staining serial sections, and clone those B cell clusters (e.g., germinal centers) which recognize the widest range of virus serotypes. Antibody-producing cells will be recovered by laser capture microdissection, and their expressed Igs will be amplified from mRNA using Ig heavy and light chain specific PCR primers, Recombinant Fabs produced by cloning these Ig fragments into expression phagemids will be studied to assay their neutralizing potency and serotype range, and to define their target epitopes.